The goal is to develop a safe, potent, monoclonal antibody (MAb) product with neutralizing activity against variola (smallpox) virus. This immunotherapeutic product primarily is intended for prevention of infection in the face of an immediate threat of smallpox attack and for treatment to suppress infection and disease in case of exposure to smallpox. The neutralizing MAbs will provide immediate immunity, which might be crucial for providing protection to individuals that are immune-compromised or at risk for smallpox vaccination. The neutralizing MAb product might also be used as a replacement for VIG (vaccinia immune globulin) in the treatment of complications of smallpox vaccination. The product will consist of a cocktail of high affinity MAbs that are directed against critically important antigens located on the surface of EEV (extracellular enveloped virions) or IMV (intracellular mature virions) forms of the virus. IMV are released after cell lysis, are stable in the environment, represent the majority of infectious particles and play a predominant role in host-to-host transmission. By contrast, EEV have an additional outer membrane, are released from infected cells, are unstable in the environment, but appear to be responsible for direct cell-to-cell and long-range virus spread within a host. The studies described in this proposal will specifically test the hypothesis that combinations of MAbs against IMV and EEV antigens will act synergistically and have greater virus neutralizing activity than MAbs directed against single antigens. The specific targets of the MAbs in this proposal are B5R and A33R (EEV-specific proteins) and A27L and L1R (IMV-specific proteins). The first aim is selection of the MAb cocktail that provides the greatest neutralizing activity in mice (vaccinia virus challenge) and in non-human primates (monkeypox virus challenge). The second aim is completion of process development for the selected MAbs. The third aim is cGMP manufacturing of the MAb cocktail drug product. The fourth aim is completion of stability, safety and toxicology studies to support the filing of an IND for a Phase I safety study in human volunteers. The project is relevant to public health in the area of biodefense. The anti-smallpox antibody product is intended to be used to prevent smallpox infection in the face of an immediate threat of smallpox attack. It is also intended to be used to suppress infection and disease in individuals who have been exposed to smallpox as a result of an attack.